Histological analysis of cells and tissues obtained, for example, from plants or animals, often involves microscopic analysis of such materials. The cells of most multicellular organisms are disposed in tissues, which can include one or more of living, dead, or inactive cells of various types, extracellular materials (e.g., intracellular matrices produced by the animal or plant), voids or spaces, and heterogenous materials (e.g., cells of other organisms, such as parasites or pathogens, or non-living materials such as mineral particles or other inclusions. Histological analysis seeks to reveal information about the three-dimensional arrangements of such elements, interactions among them, and phenomena attributable to their positions. Localization of particular tissues, cells, or subcellular components within an embedded tissue sample can be desirable for many purposes.
Analysis of three-dimensional biological samples, either in situ (e.g., as the sample occurs on or within the body of an organism) or in an extracted sample (e.g., in a tissue biopsy) is hindered by the nearness of sample components to one another and the ‘shielding’ effect of components which surround or overlie other components. That is, it can be difficult to interpret results obtained using an analytical technique from different components of a three-dimensional biological sample owing to inability to distinguish analytical results attributable to different components of the sample. A well known technique for resolving analytical signals arising from different components of a three-dimensional sample is to cut the sample into sections and to observe the results of analytical techniques applied either to an entire section (e.g., a thin slice of a three-dimensional section) or to a surface of the section (e.g., a two-dimensional surface of a halved block having a tissue sample embedded therein).
Many methods of embedding tissue samples in a medium and thereafter cutting or slicing the medium into pieces or thin sections are known. For example, many standardized protocols are known for fixing tissues in a solution (e.g., a formalin-containing solution), optionally treating the tissues (e.g., contacting the tissue with a label to identify components or with a reagent to modify the tissue, such as to decalcify bone), infusing a stable medium (e.g., paraffin or another wax or a polymer resin such as an acrylic or epoxy resin) into the tissue to form a block in which the tissue is suspended, and thereafter cutting the block (e.g., slicing thin sections from the block using a microtome to yield a plurality of substantially parallel 5-micrometer-thick slices of the block analogous to slices of a loaf of bread) to yield sections suitable for analysis (optionally after further treatment, such as with a tissue-reactive reagent or label). A skilled artisan in this field understands that a wide variety of materials, techniques, and cutting geometries can be employed, depending on the identity of the tissue being analyzed, the reactive or labeling techniques to be employed, and the detection (e.g., light or electron microscopy) technology to be used. Selections of these materials, criteria, and techniques are routine in the art and are not the subject matter to which the current disclosure is directed.
A difficulty inherent in sectioning a block having a tissue sample embedded therein is determining where the cut was made, relative to the dimensions of the block, relative to the dimensions of the embedded tissue sample, or both. For example, when a sample block is sliced into a multiplicity of slices of substantially uniform thickness, it can be difficult to determine the original position within the block to which an individual slice corresponds. By analogy, when a loaf of uniform bread of substantially constant cross section is sliced into a multiplicity of parallel slices, most or all slices of the bread appear substantially identical, and reassembling the slices into their original order can be difficult unless each slice is captured as it is cut from the loaf and the order in which the slices are cut is carefully monitored. Similar difficulty attends microtome-slicing of a paraffin-embedded tissue sample, for example, and determining the original position and conformation of each slice within the paraffin block can be difficult if the order is not carefully monitored.
Difficulties in understanding positional correspondence between material in a sectioned block and the same material in the block prior to sectioning can arise from imprecision or unpredictability in techniques used to section the block. When a block having an embedded tissue sample is to be sectioned along a plane through the block, the actual plane of cutting may be displaced from its expected position (or an expected planar cut may actually not be made in a single plane) for a number of reasons, including imprecision in the sectioning apparatus, anomalies in sectioning operation (vibration or improper operator techniques), anomalies in block properties (e.g., discontinuities in block composition or in the properties of embedded tissues), or other reasons. In many instances, such as when a multiplicity of slices of substantially uniform thickness are being made, the precise location of sectioning corresponding to each slice is not critical, so long as the position of the resulting section in the original block can be reconstructed.
In each of these instances, it can be important to be able to derive—from information available in a section of the block—a relative location of the section within the block. The “relative” location of a section can be relative to a surface of the block prior to sectioning, relative to an adjacent section of the block, relative to a single ‘reference’ section, relative to an arbitrarily fixed marker within the original block, or relative to any other indicium that permits one who analyzes the section to identify its three-dimensional location within the block prior to sectioning (or, alternatively, to identify the three-dimensional location of a detectable portion of the section within the block prior to sectioning). That is, it can be important to be able to determine where—before the block was sectioned—a section or portion thereof originated within the block, either absolutely (i.e., relative to the boundaries of the block) or relatively (i.e., relative to a selected location on or within the block). It is to this task—relating the position of an item within a sectioned block to the position at which the item existed within the not-yet-sectioned block—to which the subject matter of this disclosure relates. The process of relating positions within block sections to pre-sectioning positions within the corresponding block is sometimes referred to as three-dimensional reconstruction of the sectioned block, in that performance of these techniques can permit visualization of the original position within the block of each section thus analyzed.
Three-dimensional reconstruction of a sectioned block containing a tissue sample can be performed by image-comparison methods, whereby image-to-image registration of two dimensional images of individual sections is used to reconstruct the block. However, such techniques exhibit significant shortcomings. Registration of adjacent images can often be accomplished only if the corresponding sections are similarly stained; this can be impossible if adjacent sections (i.e., which share a common tissue) are to be differently-stained or -treated. Three dimensional tissue reconstruction is also laborious and time-consuming. Furthermore, if a gap exists between two sections increases (e.g., one or more intervening sections is lost, differently stained, or simply exhibits a different tissue pattern), registration accuracy can decrease significantly.
Others have described methods of embedding materials within tissue-embedding blocks in order to permit three-dimensional reconstruction of the original blocks.
Bussolati et al. (2005, J. Cell. Mol. Med. 9(2):438-445), for example, disclose drilling holes in a block in which a tissue is embedded and embedding cores (obtained from a different, easily identified embedded tissue) into the resulting bore holes. Sectioning of the core-embedded blocks yielded slices at which the relative positions of the embedded cores could be detected. Bussolati describe embedding cores along non-parallel axes (see, e.g., Bussolati FIG. 2) in order to permit determination of the distance between serial sections of a single block.
Similarly, Shields et al. disclose (U.S. patent application 2006/0051736) insertion of detectable cylindrical cores into a block having a tissue sample embedded therein, the insertion occurring during or after the embedding operation. The cylindrical cores described by Shields include a detectable marker within them, and detection of the marker(s) corresponding to the inserted cores can be used for three-dimensional reconstruction of a block from which multiple sections are made.
A significant shortcoming of techniques described by others for embedding detectable cylindrical cores in embedded tissue sample blocks is their impracticality. The methods described in Bussolati involve drilling and insertion of cores into already-embedded tissue samples, and Shields describes similar methods. Shields also discloses simultaneous embedding of tissue samples and detectable cores. Even if these methods could be practically performed as a part of ordinary histological embedding and analytical techniques, these methods require complicated manipulations of tissue samples and cores which may well be beyond the abilities of ordinary histology laboratory workers. It would be beneficial if a technology were available to such workers for embedding fiducial markers in blocks in which tissue samples are embedded and if that technology did not require the complicated manipulations required by methods previously described by others.
Another significant shortcoming of known tissue-embedding and -sectioning techniques relates to establishment and confirmation of the identity of sectioned samples. Blocks in which biological samples are embedded frequently look nearly identical to one another, all the more so when the embedding materials and the molds used to form the blocks are the same. Furthermore, sections taken from such blocks tend to appear nearly identical to the naked eye, and can be difficult to differentiate from one another, even when examined microscopically or following staining or labeling operations. Currently, workers must be very careful to keep detailed records of the identities of blocks and sections and to label and segregate blocks and sections sufficiently to prevent confusion or mixing of samples. Furthermore, if labels or records or lost, it can be difficult or impossible to resolve blocks or tissue samples corresponding to those labels or records. These difficulties could be reduced or avoided by a technology that reliably and enduringly associated identifying information with blocks or sections (or, preferably, both).
The following disclosure relates to such technologies.